The present invention relates to a novel composition which is useful for performing improved liquid chromatography. More particularly, the present invention relates to an improved chromatographic composition and method for performing cation-exchange chromatography where attached to the synthetic resin support particles employed therein are both (1) standard ionic cation-exchange functional groups such as sulfonates, carboxylates and/or phosphonates and (2) non-ionic crown ether-based functional groups, thereby providing a bifunctional stationary phase which provides unique separation characteristics and selectivity for numerous cationic species including alkali metals, alkaline-earth metals, ammonia, amines, and the like. The presently described compositions, therefore, provide both novel and enhanced cationic separation capabilities.
The separation of cations from a mixture of different cations is typically accomplished by cation-exchange chromatography using a cation-exchange stationary phase with ionic, acidic groups as the cation exchangers (Small, Ion Chromatography, Plenum Press, New York (1989)). Cation-exchange chromatography is a well known technique for the analysis and separation of cations in solutions wherein the technique typically includes a chromatographic separation step using an eluent solution containing an electrolyte. During the chromatographic separation step, cations of an introduced sample are eluted through a chromatography column which comprises an insoluble stationary phase to which functional cation-exchange groups are attached. Cations traversing through the column and contacting the stationary phase are then capable of exchanging at these functional cation-exchange sites. Cations which interact with the cation-exchange sites for longer periods of time elute from the chromatography column after cations which interact with those sites for shorter periods of time. For the most part, ionic acidic groups such as sulfonate, carboxylate or phosphonate groups or mixtures thereof are employed as the principle functional groups of typical cation-exchange columns.
Depending upon the type of functional group that is linked to the stationary phase of a typical cation-exchange chromatography column, different cation elution profiles are obtained. For example, standard cation-exchange chromatography columns which employ a mixture of carboxylate and phosphonate functional groups provide an elution profile where lithium elutes from the column first followed in order by sodium, ammonium, potassium, magnesium, manganese and finally calcium (Rey et al., Journal of Chromatography A 739:87-97 (1996)). However, some cationic species elute in peaks which overlap with other cationic species that elute either immediately therebefore or immediately thereafter, thereby providing a less than completely efficient separation. Moreover, when one cationic species is present at significantly higher concentrations than another cationic species, separation of the two from a mixture thereof may be very difficult. Compositions and methods which provide a further means for enhancing the separation capabilities of cation-exchange chromatography columns, therefore, would be very useful.
Crown ethers are macrocyclic polyether compounds that are capable of selectively forming complexes with a variety of different cationic species. Izatt et al., Chem. Rev. 85:271 (1985), Bajaj et al., Coord. Chem. Rev. 87:55 (1988) and Lamb et al., Journal of Chromatography 482:367-380 (1989). These compounds are referred to as "crowns" because their chemical structures resemble the shape of the regal crown and because of their ability to "crown" cationic species by complexation. The ability of a crown ether molecule to complex with a cation is dependent upon the size of the hole formed by macrocyclic structure and, as a result, crown ethers of different sizes exhibit significantly different specificities for the complexation of cations. Buschmann et al., Journal of Solution Chemistry 23(5):569-577 (1994). For example, some crown ethers readily form complexes with sodium ion but are incapable of effectively complexing with potassium ion, other crown ethers effectively complex with cesium or rubidium but not with calcium or lithium. The cation complexation characteristics of many crown ether molecules have been well documented in the literature, e.g., see Hiraoka, "Crown Ethers and Analogous Compounds", Elsevier Science Publishers, Amsterdam, (1992) and Buschmann et al., (1994) supra.
Crown ether compounds have been made part of chromatographic stationary phases and employed as cation-exchange functional groups in cation-exchange chromatography columns. Blasius et al., Journal of Chromatography 167:307-320 (1978), Delphin et al., Anal. Chem. 50(7):843-848 (1978), Lamb et al., supra, Hayashita et al., Ana. Chem. 62:2283-2287 (1990), Shirai et al., Journal of Polymer Science A: Polymer Chemistry 28:2563-2567 (1990), Hayashita et al., Anal. Chem. 63:1844-1847 (1991), Hayashita and Bartsch, Anal. Chem-. 63:1847-1850 (1991), Hiraoka, supra, Okada et al., Anal. Chem. 66:1654-1657 (1994) and Laubli et al., Journal of Chromatography A 706:103-107 (1995). However, cation-exchange resins based solely upon crown ether functional groups often exhibit poor chromatographic efficiency due to the slow rate of binding and release of the cation from the crown ether macrocycle structure and also may be too selectively "cation-specific" for many applications.
Crown ether functional groups have not previously been employed in combination with standard non-crown ether cation-exchange functional groups such as sulfonates, carboxylates or phosphonates which are independently and separately attached to a solid pahse. The combination of standard cation-exchange resins used in ion chromatography with the attachment of functional crown ethers to synthetic resin support particles, thereby resulting in bifunctional cation-exchange resins, is provided herein. These bifunctional resins provide novel cation separation capacity.